Ectomycorrhizae of Jack Pine and Green Alder: Assessment for the Need for Innoculation, Development of Innoculation Techniques, and Outplanting Trials on Oil Sands Tailings

The overall objectives of these studies were to characterize the mycorrhizal status of jack pine and green alder which are prime candidates as reclamation species for oil sand tailings and to determine the potential benefits of mycorrhizae on plant performance. This entailed determining the symbiont status of container-grown nursery stock and the quantity and quality of inoculum in reconstructed soils, developing inoculation techniques and finally, performance testing in an actual reclamation setting. Much emphasis has been placed on gaining information on the fungi involved in the symbioses as little is known about the ecology of mycorrhizal fungi and this information is essential for successfully applying the technology. Seedlings from seven nurseries in Alberta and British Columbia were examined for mycorrhizae. The conversion of short root to mycorrhizae varied from 0 to 100% depending upon the particular nursery, fertilizer regimes, and residence time in the nursery. It is without doubt that many sites are being planted with nonmycorrhizal jack pines, lodgepole pine, and white spruce. The most common fungi in the infested nurseries were E-strain, Thelephora terrestris and Mycelium radicis atrovirens. Amphinema byssoides occurred exclusively on spruce and succeeded E-strain and Thelephora in the nursery. Coltricia perennis, a polypore, was found on nursery stock for the first time. In one nursery, E-strain appeared to prevent the development of a seedling disease .caused by a species of Cylindrocarpon. Alder seedlings from three nurseries were all nonmycorrhiza1 but most seedlings were nodulated. The second major source of natural inoculum is the reconstructed soils and the inoculum source within the soils is the muskeg peat. Inoculum in undisturbed and stockpiled peat was assayed by using a greenhouse baiting technique and jack pine seedlings. The most common fungi were E-strain, I-type (Tuber sp.), and a hyaline Basidiomycete. Stockpiling the peat reduced the infectivity of the peat and plants were still mycorrhizal deficient after 4 months in the greenhouse. Additional experiments showed that the greenhouse baiting technique accurately reflected the species that develop in the field but over estimates the rates of mycorrhi zation. The reconstructed soi1 contained adequate quantities of Frankia inoculum to nodulate alder but mycorrhizal inoculum compatible with alder was present only in very small quantities. From the nursery and indigenous inoculum studies it could be concluded that both jack pine and green alder planted on oil sand tailings amended with muskeg peat would potentially be deficient in mycorrhizae and this deficiency could adversely affect plant performance. In order to determine if mycorrhizae would benefit plant performance in the field, two outplanting trials were conducted; the first with jack pine and the second with jack pine and green alder. The second trial was limited in scope due to heavy winter mortality soon after planting. In the first trial, container-grown jack pine seedlings were inoculated with 12 fungi and mycorrhizae were formed by 9 of the 12 species. The most aggressive fungi (those producing the highest levels of short root infections) were Thelephora terrestris, Laccaria proxima, Hebeloma sp. and E-strain, all of which are known as \"weedy\" nursery species. A lower degree of infection was achieved with Cenococcum geophilum, Pisolithus tinctorius, Astraeus hygrometricus, Lactarius paradoxus, and Sphaerospore 11 a brunnea. Amphi nema byssoides, Hydnum imbricatum, and Tricholoma flavovirens failed to form any mycorrhizae. All the above inoculation treatments plus two uninoculated controls, one grown with the inoculated seedlings and the other from the Syncrude greenhouse, were outplanted in the spring on the Syncrude dyke. The reconstructed soil consisted of extracted oil sands, muskeg peat, and clayey overburden. After one season in the field, T. terrestris, L. proxima, Hebeloma sp., and E-strain had all readily infected the new roots that extended into the reconstructed soi1. The other fungi were poor colonizers of jack pine roots in the field. Competition from indigenous fungi was not a factor in the degree of success as only 4% of the short roots were infected by indigenous species. Growth of jack pine was not significantly affected by the presence of mycorrhizae during the first growing season. Laccaria proxima completely disappeared after 1 year and between the second and third year, Hebel oma sp. and The1ephora terrestris almost completely disappeared. Of the introduced fungi, only E-strain was present in substantial quantities after 3 years and it appeared also to be disappearing. It appeared that the major replacement process was noninteractive, i.e., the resident fungi died, and the roots were subsequently reinfected by another fungus. The colonization by indigenous fungi increased each year, rising to 33% in the second year and to 72% by the end of the third year. The most common indigenous fungi were E-strain, I-type (Tuber sp.), Mycelium radicis atrovirens, a Rhizopogon-like fungus, and a hyaline Basidiomycete. The latter four species increased with time, whereas E-strain appeared to be decreasing in abundance after 3 years. Shoot weights of seedlings inoculated with E-strain and Thelephora terrestris were 2- to 3-fold larger than the controls after 2 years growth but the differences in size decreased in the third year. In the second outplanting study, inoculation could not be assessed due to heavy mortality but it was observed that three E-strain fungi and an I-type isolate all readily colonized new roots in the reconstructed soils. Alder lacked both nodules and mycorrhizae when planted but all plants became nodulated in the first year. However, mycorrhizal development was still often poor even after 2 years in the field. The major fungus associated with alder was Alpova diplophoeus which was also the dominant fungus on naturally regenerating plants. In the jack pine outplanting study fertilizer was used conservatively in the greenhouse phase so as to ensure maximum mycorrhizal development. Consequently, the seedlings were small and thus some losses were encountered in the field due to flooding of low areas of the experimental plot. Subsequent fertilizer trials have demonstrated that seedling size need not be sacrificed for successful mycorrhizal development when certain, aggressive fungi are used. The most fertilizer tolerant fungus tested was an E-strain isolate. All short roots were ectomycorrhizal when fertilizer containing 60 mg N L-1 was applied three times weekly. This rate approaches that used in commercial operations. Even when the rate was doubled, E-strain still infected a substantial portion of the short roots. Hebeloma sp. And Lactarius paradoxus were more sensitive to fertilizer levels infecting 20-30% of the short roots at the 60 mg N L-1 1eve1. Astraeus hygrometricus and Amphinema byssoides did not form ectomycorrhizae at this level, however this may have been due to an overall low inoculums potential of these two species. It is proposed that host resistance is elevated by high fertilizer levels and that it may be possible to overcome this resistance by increasing inoculum potential and thus, to expand the taxonomic spectrum of fungi that can be successfully introduced onto container-grown seedlings. A simple change from using an organic growing medium to an inorganic medium did not result in any ectomycorrhizal formation by two fungi which continue to resist artificial inoculation attempts, Amphinema byssoides and Suillus tomentosus. As these fungi appear to persist through several succession stages (multi-stage fungi) they may be more desirable than species currently being tested such as E-strain, Laccaria proxima and Thelephora terrestris which are early-stage (pioneer) fungi. One group of ectomycorrhizal fungi not included in any inoculation program are those species occurring in the late successional stages of a forest stand (late-stage fungi). At this point mycorrhizal infections cannot be initiated from either spores or mycelium. However, at least some of these fungi can be very aggressive when spreading from established ectomycorrhizae to nonmycorrhizal seedlings. Nothing is known of the relative benefits on plant performance of these late-stage fungi versus the early-stage fungi whose use is currently in vogue. It appears that multi- and late-stage fungi are more sensitive to certain biological soil factors than early-stage fungi. Preliminary attempts to reduce fungal antagonism by treating planting mixtures with the fungicide benomyl was unsuccessful. However, fly maggots which were observed consuming inoculum, were eliminated by drenching the planting mixtures with Oiazinon. By using intact cultures of fungi it was possible to increase inoculum potential and to observe consumption and colonization of the inoculum. Evidence from ~ series of trials indicate that if the antagonistic mold, Trichoderma harzianum and fungal feeding fly larvae can be controlled, it may be possible to inoculate many multi- and late-stage fungi and test efficiencies in the field. Attempts to inoculate green alder with pure cultures of Frankia and Alpova diplophloeus failed, however, a soil inoculum was used successively to 'promote both nodulation and mycorrhization. It was necessary to reduce fertilizer levels to concentrations well below those used commercially to obtain mycorrhizal infections. Field-collected nodules also served as a good inoculum source for Frankia. Although antagonistic fungi and maggots could be controlled with pesticides, bacteria rapidly colonized inoculum of Alpova and killed the fungus. Further work is necessary to improve inoculation procedures so a wider range of fungi can be introduced onto ectomycorrhizal hosts and be subsequently tested for efficacy on reclamation sites.

This material is provided under educational reproduction permissions included in Alberta Environment and Sustainable Resource Development's Copyright and Disclosure Statement, see terms at http://www.environment.alberta.ca/copyright.html. This Statement requires the following identification: \"The source of the materials is Alberta Environment and Sustainable Resource Development http://www.environment.gov.ab.ca/. The use of these materials by the end user is done without any affiliation with or endorsement by the Government of Alberta. Reliance upon the end user's use of these materials is at the risk of the end user.